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International Journal of Biological... Jun 2024With multiscale hierarchical structure, wood is suitable for a range of high-value applications, especially as a chromatographic matrix. Here, we have aimed to provide a...
With multiscale hierarchical structure, wood is suitable for a range of high-value applications, especially as a chromatographic matrix. Here, we have aimed to provide a weak anion-exchange polymeric monolithic column based on natural wood with high permeability and stability for effectively separating the targeted protein. The wood-polymeric monolithic column was synthesized by in situ polymerization of glycidyl methacrylate and ethylene glycol dimethacrylate in wood, and coupled with diethylaminoethyl hydrochloride. The wood-polymeric monolithic column can be integrated with fast-protein liquid chromatography for large-scale protein purification. According to the results, the wood-polymeric monolithic column showed high hydrophilicity, permeability and stability. Separation experiments verified that the wood-polymeric monolithic column could purify the targeted protein (spike protein of SARS-COV-2 and ovalbumin) from the mixed proteins by ion exchange, and the static adsorption capacity was 33.04 mg mL and the dynamic adsorption capacity was 24.51 mg mL. In addition, the wood-polymerized monolithic column had good stability, and a negligible decrease in the dynamic adsorption capacity after 20 cycles. This wood-polymerized monolithic column can provide a novel, efficient, and green matrix for monolithic chromatographic columns.
Topics: Wood; Adsorption; Methacrylates; Chromatography, Ion Exchange; Polymers; Ovalbumin; Hydrophobic and Hydrophilic Interactions; SARS-CoV-2; Polymerization; Epoxy Compounds
PubMed: 38740162
DOI: 10.1016/j.ijbiomac.2024.132310 -
Methods in Molecular Biology (Clifton,... 2024Cell-surface receptors can be difficult to express and purify for structural and biochemical studies due to low expression levels, misfolding, aggregation, and...
Cell-surface receptors can be difficult to express and purify for structural and biochemical studies due to low expression levels, misfolding, aggregation, and instability. Cell-surface receptor ectodomains are more amenable to large-scale production, but this requires designing and testing various truncation constructs. However, since each protein is unique, testing these constructs individually for many targets is a time-consuming process. In this context, we present a high-throughput ELISA fluorescence approach that allows the rapid assessment of numerous recombinant constructs simultaneously. Cell-surface ectodomains are expressed in small scale, enzymatically biotinylated, and detected using a C-terminal His-tag. As an example, we tested the expression of truncation constructs for the neurexin, neuroligin, and latrophilin families and show that the small-scale ELISA allowed us to prioritize well-expressing construct for large-scale production. By employing this method, one can efficiently detect clones with low expression levels, streamlining the process and saving valuable time in identifying optimal candidates for further study.
Topics: Humans; Enzyme-Linked Immunosorbent Assay; High-Throughput Screening Assays; Membrane Proteins; Protein Domains; Recombinant Proteins; HEK293 Cells; Gene Expression
PubMed: 38926287
DOI: 10.1007/978-1-0716-3878-1_19 -
The Protein Journal Apr 2024Scavenger receptors are a protein superfamily that typically consists of one or more repeats of the scavenger receptor cysteine-rich structural domain (SRCRD), which is...
Scavenger receptors are a protein superfamily that typically consists of one or more repeats of the scavenger receptor cysteine-rich structural domain (SRCRD), which is an ancient and highly conserved protein module. The expression and purification of eukaryotic proteins containing multiple disulfide bonds has always been challenging. The expression systems that are commonly used to express SRCRD proteins mainly consist of eukaryotic protein expression systems. Herein, we established a high-level expression strategy of a Type B SRCRD unit from human salivary agglutinin using the Escherichia coli expression system, followed by a refolding and purification process. The untagged recombinant SRCRD was expressed in E. coli using the pET-32a vector, which was followed by a refolding process using the GSH/GSSG redox system. The SRCRD expressed in E. coli SHuffle T7 showed better solubility after refolding than that expressed in E. coli BL21(DE3), suggesting the importance of the disulfide bond content prior to refolding. The quality of the refolded protein was finally assessed using crystallization and crystal structure analysis. As proteins refolded from inclusion bodies exhibit a high crystal quality and reproducibility, this method is considered a reliable strategy for SRCRD protein expression and purification. To further confirm the structural integrity of the refolded SRCRD protein, the purified protein was subjected to crystallization using sitting-drop vapor diffusion method. The obtained crystals of SRCRD diffracted X-rays to a resolution of 1.47 Å. The solved crystal structure appeared to be highly conserved, with four disulfide bonds appropriately formed. The surface charge distribution of homologous SRCRD proteins indicates that the negatively charged region at the surface is associated with their calcium-dependent ligand recognition. These results suggest that a high-quality SRCRD protein expressed by E. coli SHuffle T7 can be successfully folded and purified, providing new options for the expression of members of the scavenger receptor superfamily.
Topics: Escherichia coli; Humans; Crystallography, X-Ray; Protein Refolding; Recombinant Proteins; Crystallization; Agglutinins; Protein Domains; Gene Expression; Models, Molecular; Cysteine; Receptors, Scavenger
PubMed: 38265733
DOI: 10.1007/s10930-023-10173-x -
Food Chemistry Aug 2024This study presents a novel approach toward the one-pot green synthesis of ZIF-8/IgG composite, focusing on its precise orientation and protection of the anti-aflatoxins...
One-pot green synthesis of ZIF-8/IgG composite for the precise orientation and protection of antibody and its application in purification and detection of aflatoxins in peanut oil.
This study presents a novel approach toward the one-pot green synthesis of ZIF-8/IgG composite, focusing on its precise orientation and protection of the anti-aflatoxins antibody. The antibody orientation is achieved through the specific binding of IgG to the Fc region of the antibody, while the antibody protection is accomplished by the structural change restriction of ZIF-8 framework to the antibody. Consequently, the antibody exhibits enhanced target capability and significantly improved tolerance to organic solvents. The ZIF-8/IgG/anti-AFT was employed for the purification and detection of AFTs by coupling with UPLC. Under optimized conditions, the recoveries of spiked AFTs in peanut oils are between 86.1% and 106.4%, with relative standard deviations (RSDs) ranging from 0.8% to 8.8%. The linearity range is 0.5-20.0 ng for AFB and AFG, 0.125-5.0 ng for AFB and AFG, the limit of detection is 0.1 ng for AFB and AFG, 0.03 ng for AFB and AFG.
Topics: Aflatoxins; Food Contamination; Peanut Oil; Green Chemistry Technology; Immunoglobulin G; Antibodies; Chromatography, High Pressure Liquid
PubMed: 38604030
DOI: 10.1016/j.foodchem.2024.139272 -
Revista Internacional de Andrologia Mar 2024It is estimated that microorganisms colonize 90% of the body surface. In some tracts, such as the genitourinary tract, the microbiota varies throughout life, influenced...
It is estimated that microorganisms colonize 90% of the body surface. In some tracts, such as the genitourinary tract, the microbiota varies throughout life, influenced by hormonal stimulation and sexual practices. This study evaluated the semen differences and presence of , , and in semen samples from patients with symptoms of chronic prostatitis and men asymptomatic for urogenital infections. Fifty-three semen samples were included: 22 samples from men with symptoms of chronic prostatitis and 31 asymptomatic men (control group). In addition to the presence of , , and , semen parameters, total antioxidant capacity of seminal plasma, prostatic antigen and some proinflammatory cytokines were evaluated in each semen sample. Volunteers with symptoms of chronic prostatitis presented a lower percentage of sperm morphology (4.3% control group 6.0%, = 0.004); in the semen samples of volunteers in the group asymptomatic for urogenital infections, microorganisms associated with the vaginal microbiota were detected more frequently. The presence of bacteria in the vaginal microbiota can also benefit male reproductive health, which undergoes various modifications related to lifestyle habits that are susceptible to modification. Microorganisms associated with the vaginal microbiota, such as , , and , may have a protective role against the development of male genitourinary diseases such as prostatitis.
Topics: Humans; Male; Prostatitis; Semen; Adult; Microbiota; Coitus; Gardnerella vaginalis; Lactobacillus; Vagina; Middle Aged; Actinobacteria; Female; Young Adult; Chronic Disease; Case-Control Studies; Semen Analysis; Cytokines
PubMed: 38735876
DOI: 10.22514/j.androl.2024.006 -
Angewandte Chemie (International Ed. in... Dec 2023DNA repair protein RAD51 is a key player in the homologous recombination pathway. Upon DNA damage, RAD51 is transported into the nucleus by BRCA2, where it can repair...
DNA repair protein RAD51 is a key player in the homologous recombination pathway. Upon DNA damage, RAD51 is transported into the nucleus by BRCA2, where it can repair DNA double-strand breaks. Due to the structural complexity and dynamics, researchers have not yet clarified the mechanistic details of every step of RAD51 recruitment and DNA repair. RAD51 possesses an intrinsic tendency to form oligomeric structures, which make it challenging to conduct biochemical and biophysical investigations. Here, for the first time, we report on the isolation and characterization of a human monomeric RAD51 recombinant form, obtained through a double mutation, which preserves the protein's integrity and functionality. We investigated different buffers to identify the most suitable condition needed to definitively stabilize the monomer. The monomer of human RAD51 provides the community with a unique biological tool for investigating RAD51-mediated homologous recombination, and paves the way for more reliable structural, mechanistic, and drug discovery studies.
Topics: Humans; DNA Damage; DNA Repair; Homologous Recombination; Neoplasms; Rad51 Recombinase; Mutation; Protein Stability; Protein Domains; Recombinant Proteins
PubMed: 37924230
DOI: 10.1002/anie.202312517 -
International Journal of Biological... Jun 2024As the rapid and accurate screening of infectious diseases can provide meaningful information for outbreak prevention and control, as well as owing to the existing... (Review)
Review
As the rapid and accurate screening of infectious diseases can provide meaningful information for outbreak prevention and control, as well as owing to the existing limitations of the polymerase chain reaction (PCR), it is imperative to have new and validated detection techniques for SARS-CoV-2. Therefore, the rationale for outlining the techniques used to detect SARS-CoV-2 proteins and performing a comprehensive comparison to serve as a practical benchmark for future identification of similar viral proteins is clear. This review highlights the urgent need to strengthen pandemic preparedness by emphasizing the importance of integrated measures. These include improved tools for pathogen characterization, optimized societal precautions, the establishment of early warning systems, and the deployment of highly sensitive diagnostics for effective surveillance, triage, and resource management. Additionally, with an improved understanding of the virus' protein structure, considerable advances in targeted detection, treatment, and prevention strategies are expected to greatly improve our ability to respond to future outbreaks.
Topics: SARS-CoV-2; Humans; COVID-19; Viral Proteins
PubMed: 38734351
DOI: 10.1016/j.ijbiomac.2024.132237 -
The Journal of Biological Chemistry Sep 2023Reductive dehalogenases are corrinoid and iron-sulfur cluster-containing enzymes that catalyze the reductive removal of a halogen atom. The oxygen-sensitive and...
Reductive dehalogenases are corrinoid and iron-sulfur cluster-containing enzymes that catalyze the reductive removal of a halogen atom. The oxygen-sensitive and membrane-associated nature of the respiratory reductive dehalogenases has hindered their detailed kinetic study. In contrast, the evolutionarily related catabolic reductive dehalogenases are oxygen tolerant, with those that are naturally fused to a reductase domain with similarity to phthalate dioxygenase presenting attractive targets for further study. We present efficient heterologous expression of a self-sufficient catabolic reductive dehalogenase from Jhaorihella thermophila in Escherichia coli. Combining the use of maltose-binding protein as a solubility-enhancing tag with the btuCEDFB cobalamin uptake system affords up to 40% cobalamin occupancy and a full complement of iron-sulfur clusters. The enzyme is able to efficiently perform NADPH-dependent dehalogenation of brominated and iodinated phenolic compounds, including the flame retardant tetrabromobisphenol, under both anaerobic and aerobic conditions. NADPH consumption is tightly coupled to product formation. Surprisingly, corresponding chlorinated compounds only act as competitive inhibitors. Electron paramagnetic resonance spectroscopy reveals loss of the Co(II) signal observed in the resting state of the enzyme under steady-state conditions, suggesting accumulation of Co(I)/(III) species prior to the rate-limiting step. In vivo reductive debromination activity is readily observed, and when the enzyme is expressed in E. coli strain W, supports growth on 3-bromo-4-hydroxyphenylacetic as a sole carbon source. This demonstrates the potential for catabolic reductive dehalogenases for future application in bioremediation.
Topics: Escherichia coli; NADP; Oxygen; Vitamin B 12; Phenols; Electron Spin Resonance Spectroscopy; Hydrolases; Rhodobacteraceae; Protein Structure, Tertiary; Models, Molecular; Maltose-Binding Proteins; Recombinant Fusion Proteins; Coenzymes
PubMed: 37495113
DOI: 10.1016/j.jbc.2023.105086 -
Journal of Biochemistry Apr 2024The ribosome, the protein synthesizing machinery composed of dozens of proteins and several ribosomal RNAs (rRNAs), is essential for life. In vitro reconstitution of the...
The ribosome, the protein synthesizing machinery composed of dozens of proteins and several ribosomal RNAs (rRNAs), is essential for life. In vitro reconstitution of the ribosome holds significance for understanding biosynthesis, applications in biotechnology and potential contributions to synthetic biology. There is a long history of in vitro reconstitution of bacterial ribosomes, originating in the 1970s when the 30S ribosome of Escherichia coli was reconstituted from the protein and rRNA components prepared from native ribosome. Since then, the reconstitution using in vitro transcribed rRNAs has been established, and more recently, the reconstitution using recombinant ribosomal proteins has also become possible. A recent report by Aoyama et al. (J. Biochem. 2022; 171:227-237), the reconstitution of the 50S ribosome using 33 recombinant ribosomal proteins, is a new leap toward complete reconstitution of the holo ribosome complex from recombinant proteins and in vitro transcribed rRNAs. This commentary also discusses future challenges.
Topics: Escherichia coli; Escherichia coli Proteins; Recombinant Proteins; Ribosomal Proteins; Ribosomes; RNA, Ribosomal
PubMed: 38236695
DOI: 10.1093/jb/mvad121 -
Journal of Bioscience and Bioengineering Jul 2024A new extracellular protease from Bacillus subtilis strain MPK with collagenolytic activity was isolated and purified. Fish skin which otherwise would be treated as...
A new extracellular protease from Bacillus subtilis strain MPK with collagenolytic activity was isolated and purified. Fish skin which otherwise would be treated as waste is used as substrate for the production of protease. Using various techniques such as ammonium sulphate precipitation and ion exchange chromatography, protease was purified and characterized subsequently. Protease of approximately 61 kDa molecular weight was purified by 135.7-fold with 18.42% enzyme recovery. The protease showed effective properties like pH and temperature stability over a broad range with optimum pH 7.5 and temperature 60 °C. K and V were found to be 1.92 mg ml and 1.02 × 10 mol L min, respectively. The protease exhibited stability in various ions, surfactants, inhibitors and organic solvents. Subsequently, the protease was successfully utilized for collagen hydrolysis to generate collagen peptides; thus, the produced protease would be a potential candidate for multifaceted applications in food and pharmaceutical industries due to its significant characteristics and collagenolytic properties.
Topics: Bacillus subtilis; Collagen; Hydrogen-Ion Concentration; Temperature; Hydrolysis; Molecular Weight; Enzyme Stability; Animals; Peptide Hydrolases; Bacterial Proteins; Skin; Fishes; Chromatography, Ion Exchange
PubMed: 38637241
DOI: 10.1016/j.jbiosc.2024.03.003